In Vitro 5-Lipoxygenase Inhibition of Polyphenolic Antioxidants from Undomesticated Plants of South Africa
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Journal of Medicinal Plants Research Vol. 2(9), pp. 207-212, September, 2008 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875© 2008 Academic Journals Full Length Research Paper In vitro 5-Lipoxygenase inhibition of polyphenolic antioxidants from undomesticated plants of South Africa Uma Sankar Akula and Bharti Odhav* Durban University of Technology, Faculty of Engineering, Science and the Built Environment, Department of Biotechnology, ML Sultan Campus, P.O. Box 1334, Durban, 4000, South Africa. Accepted 10 July 2008 Aqueous and methanolic extracts of 18 leafy vegetables from South Africa were investigated for their free radical scavenging activity, total phenolic content and anti-inflammatory properties. Amaranthus dubius, Amaranthus hybridus, Amaranthus spinosus, Asystasia gangetica, Bidens pilosa, Centella asiatica, Ceratotheca triloba, Chenopodium album, Cleome monophylla, Emex australis, Galinsoga parviflora, Justicia flava, Momordica balsamina, Oxygonum sinuatum, Physalis viscosa, Portulaca oleracea, Senna occidentalis and Solanum nigrum were used in this study. In the radical scavenging studies using 2,2-diphenyl-2-picrylhydrazyl hydrate(DPPH), methanolic extracts from six plants almost completely inhibited DPPH absorption (J. flava-96.6%, P. oleracea-96.5%, M. balsamina-94.7%, O. sinuatum-92.1%, S. nigrum-92.0% and A. hybridus-90.5%), whereas methanolic extracts from E. australis-78.6% and G. parviflora-76.5% showed lowest activities among the plants studied. Aqueous extracts were considerably less effective radical scavengers compared to methanolic extracts showing highest activity with J. flava-48.0% and lowest with A. gangetica-36.2%. A correlation between radical scavenging activity of extracts with total phenolic content was observed. Out of 18 plants studied for anti-inflammatory activity, methanolic extracts from eight plants showed significant inhibition of 5- lipoxygenase (5-Lox) activity. Among the plants studied Bidens pilosa (IC50 21.8 µg/ml) showed maximum anti-inflammatory activity while Emex australis (IC50 81.4 µg/ml) showed minimum inhibition of 5-Lox activity. Large amounts of phenolic compounds may contribute towards the antioxidant and anti-inflammatory properties among the plants studied. Key words: Leafy vegetables, radical scavenging, DPPH, total phenolics, 5-lipoxygenase, anti-inflammatory. INTRODUCTION The uncontrolled production of oxygen free radicals and of ageing, such as cancer, cardiovascular disease, cata- the unbalanced mechanism of antioxidant protection racts, immune system decline and brain dysfunction result in the onset of many diseases and accelerate age- (Ames et al., 1990; Percival, 1998; Young and Woodside, ing. The antioxidants when present at low concentrations 2001). Fortunately, free radical formation is controlled compared to that of an oxidizable substrate, significantly naturally by various beneficial compounds known as anti- delays or inhibits the oxidation of that substrate (Percival, oxidants (Percival, 1998). 1998; Young and Woodside, 2001; Atoui et al., 2005). When the availability of antioxidants is limited, this The physiological role of free radical and hydroxyl free damage can become cumulative and debilitating oxide- radical-scavengers is to prevent damage to cellular com- tive stress results. Antioxidants are capable of stabilizing, ponents arising as a consequence of chemical reactions or deactivating free radicals before the latter attack cells involving free radicals. The key role for free radicals is as and biological targets. They are therefore critical for major contributors to ageing and to degenerative diseases maintaining optimal cellular and systemic health and well- being (Percival, 1998). Most antioxidants isolated from higher plants are polyphenols, which show biological acti- *Corresponding author. E-mail: [email protected]. Tel: +27 31 vity as antibacterial, anti-carcinogenic, anti-inflammatory, 373 5330. Fax: +27 31 373 5351. anti-viral, anti-allergic, estrogenic and immune-stimulating 208 J. Med. Plant. Res. Ing effects (Larson, 1988). matory activity of a drug (Alitonou et al., 2006). The antioxidant activity of phenolics is mainly due to Many of the plant anti-oxidants are polyphenols which their redox properties, which allow them to act as reduc- have carbon-based aromatic phenyl-ring compounds ing agents, hydrogen donors, and singlet oxygen quen- which are easily oxidized to quinones by reactive oxygen chers. In addition, they have a metal chelation potential. species, a property that helps account for their free radi- Synthetic anti-oxidants such as butylated hydroxyanisole cal scavenging capacity. The leaves of many traditional (BHA), butylated hydroxytoluene (BHT) and tertbutyl- leafy vegetables supplement the diets, which consist hydroquinone (TBHQ) have been used widely as anti- mainly of cereals and grains (Van Wyk and Gericke, oxidants in foods, but concerns over the safety of use 2000). Most of these plants lack comprehensive data that have led towards interests in natural anti-oxidants can corroborate the traditional knowledge. The aim of this (Wanasundara and Shahidi, 1998). These synthetic anti- work was to investigate 18 traditional leafy vegetables oxidants are substituted phenolic compounds, and subse- found in the Kwa-Zulu Natal province of South Africa for quently much of the research on natural anti-oxidants has their anti-oxidative and anti-inflammatory potential, and also focused on phenolic compounds, in particular the correlate this to total phenolic compounds. flavonoids and hydroxycinnamic acids (Martı´nez- Valverde et al., 2002). Phenolic compounds also possess an array of potentially beneficial lipoxygenase inhibitory MATERIALS AND METHODS and anti-oxidant properties; they have been used for the Plant material treatment of inflammatory diseases (Sreejayan and Rao, 1996). Eighteen traditional leafy vegetables were used in this study. They The mechanisms of inflammation involve a series of were collected from different locations in Kwa-Zulu Natal, South events in which the metabolism of arachidonic acid plays Africa. Table 1 lists the leafy vegetables and consumption practices an important role. Prostaglandins are involved in the used in this study with the following information: scientific name, complex process of inflammation and are responsible for family, English and Zulu names. Local floristic keys were used for determining the species. Data on different species and the Zulu the pain (Jager et al., 1996). It can be metabolized by the names have been sourced from publications on the flora of South cyclooxygenase (COX) pathway to prostaglandins and Africa (Odhav et al., 2007; Hutchings, 1996). Voucher specimens thromboxane A2, or by the lipoxygenase (LOX) pathway are housed in the Ward Herbarium, University of Kwa-Zulu-Natal, to hydroperoxy-eicosatetraenoic acids (HPETE’s) and Westville campus, Durban, South Africa. leukotrienes (LT’s), which are important biologically active mediators in a variety of inflammatory events Sample extraction (Alitonou et al., 2006, Piper et al., 1994). Upon appro- priate stimulation of neutrophils, arachidonic acid is The leaves (100 g) were dried at 30ºC in a oven (Memmert) and cleaved from membrane phospholipids and can be con- powdered in a Salton blender. Ten grams were dissolved in 200 ml verted to leukotrienes and prostaglandins through 5- of dd H2O/ 80% methanol (vol/vol) for one hour and filtered with a Whatman No. 1 filter paper. The filtrate was then concentrated to lipoxygenase(5-Lox) or cyclooxygenase(Cox) pathways 15 ml using a Buchi RE 121 Roto-evaporator including a Buchi 461 respectively (Bouriche et al., 2005). Furthermore, inflam- water bath set at a temperature of 50ºC. This concentrate was matory processes also involve reactive oxygen species freeze dried using a Virtis Benchtop Freeze drier. started by leucocyte activation. Grabmann et al. (2000) found that eucalyptus and myrtle essential oils attenuated DPPH radical scavenging assay leucocyte activation by scavenging hydroxyl radicals indirectly produced by leucocyte degranulation, thereby The radical scavenging activity of plant extracts was measured interfering with inflammatory processes by acting as using the stable free radical scavenger, DPPH (2, 2-diphenyl-2- antioxidants. Therefore, screening of antioxidant pro- picrylhydrazyl hydrate), decoloration assay described by Choi perties may provide important information about the (2002). The freeze dried plant samples (1.0 mg/ml) from each of the plant extracts were diluted in ethanol to give final concentrations of potential activity of a drug on inflammatory processes 500, 250, 100, 50 and 10 µg/ ml. One ml of a 0.3 mM DPPH in (Njenga and Viljoen, 2006) The establishment of new in ethanol solution was added to 2.5 ml of sample solution of different vitro test systems has stimulated the screening of plants concentrations and allowed to react at room temperature for 30 aiming to find leads for the development of new drugs. min. The resulting colour was measured at A518 spectropho- The plant lipoxygenase pathway is in many respects the metrically (Varion Cary I-E UV visible spectrophotometer). The equivalent of the ‘arachidonic acid cascades’ in animals radical scavenging activity was measured as the decolourization percentage of the test sample using the following formula: (Gardner, 1991). For this reason, the in vitro inhibition of soybean lipoxygenase constitutes a good model for the DPPH Scavenging capacity % =100 - [(Absorbance